More than 1.8 million people die annually from infection with Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis. Current vaccines or chemotherapy regimens are not able to reverse this trend. Our long-term goal is to better understand the molecular biology of M.tb and to discover new mechanisms of survival employed by this successful pathogen. An important aspect of the host-pathogen interactions is the regulation of the acquisition of metal elements in the microenvironment where tuberculous bacilli live. The ultimate goal in this project is to identify the role of metal-responsive genes to the overall basic biology of M.tb and to examine their impact on mycobacterial phagosome maturation. Preliminary data gathered so far laid the foundation for strong hypotheses related to Cu trafficking systems. Additionally, it directed our attention to other metal ions that could be equally or even more important than Cu and Fe. Fortunately, we embarked on a new technology for Scanning Transmission Electron Microscopy (STEM) for elemental analysis of biological tissues on the ionic level that could analyze multiple ions (e.g. 10 elements) of the same sample. We decided to utilize the R21 mechanism of funding to explore the advantages of this technology and to apply it towards the better understanding of the molecular pathogenesis of M.tb. We believe that our studies will open the door for more detailed investigations of the role of specific metals in M.tb survival strategies. In the first aim of this project we will utilize the power of analytical electron microscopy to profile the elemental map in lungs of mice representing 2 different models of murine tuberculosis (BALB/c and SCID). In the second aim, we will profile the mycobacterial responses to key ions significantly changed their levels during progression from early to chronic tuberculosis. Finally, in the third aim, we will examine mycobacterial phagosome maturation following infection with bacilli defective in metal ion acquisition systems. Overall, we will take full advantage of the high resolution, micro- analytical system (STEM) and DNA microarrays to dissect the contribution of metalloproteins to metal homeostasis and to the intracellular environment of M. tb. We believe that the outcomes of this project will delineate novel mechanisms of pathogenesis and could help in designing better drugs/vaccines against tuberculosis. PUBLIC HEALTH RELEVANCE: This project is very relevant to the mission of the NIH. Tuberculosis causes a tremendous risk to healthy populations in the USA as well as immunocompromised individuals.